The transthyretin (TTR) amyloidoses are fatal disorders of secondary protein structure characterized by extracellular deposition of aggregates, including amyloid fibrils, derived from the secreted transthyretin protein. Aging is a risk factor for both the senile systemic (normal sequence) and familial (mutated sequence) forms of these disorders. At this time, no pharmacologic treatment is available. Recent data suggest that oligomers generated early in the course of TTR misfolding may be critical in the in vivo pathogenesis. We have developed the cell and molecular biologic tools to precisely analyze the molecular mechanisms underlying cell damage. Such knowledge is critical to the long-term goal of finding new therapeutic strategies to treat these diseases. Our working hypotheses are 1) TTR cytotoxicity in tissue culture reflects the human pathology regarding the susceptibility of target tissues and the amyloidogenicity of different TTR variants. 2) There is a relationship between the age-related increase in protein oxidation and the onset of TTR amyloidoses. 3) The interaction between the appropiate TTR conformer and target cells results in the generation of reactive oxygen species which, in turn, trigger an apoptotic cascade leading to cell death. 4) There is a cell receptor mediating the cytotoxic response. 5) The sequence of events leading to cell death can be prevented with compounds structurally derived from the plant polyphenol resveratrol and other antioxidants. These hypotheses will be tested in our 3 specific aims. In aim 1 we will establish tissue culture systems that mimic the human pathology in terms of tissue specificity and TTR variant. With these models in hand we will precisely identify the responsible TTR cytotoxic species, and define the role of protein oxidation and of the extracellular matrix on TTR-induced cytotoxicity. In aim 2 we will characterize and identify the cell receptor involved in the cytotoxic response using biochemical, immunochemical and proteomic mass spectrometry. In aim 3 we will use the cell system to screen compounds that inhibit TTR-induced cytotoxicity to find substances that may prevent TTR amyloidosis in vivo. We will define the mechanism of inhibition of TTR- induced cytotoxicity by the active small molecules which might in turn, be applicable to other amyloidoses. The insights gained from these studies will provide lead compounds and potential new pathways for therapeutic intervention. PUBLIC HEALTH RELEVANCE: The transthyretin amyloidoses are a set of age-related, mostly untreatable, poorly understood and ultimately fatal diseases affecting millions in the US and many more worldwide. We will create relevant tissue culture models to define the cellular mechanism of transthyretin damage and to find new compounds that can be used to treat these disorders. The findings will open new avenues for therapeutic intervention such as blockade of the formation of transthyretin toxic species, or inhibition of the cellular pathways that lead to cell death.